A master mix is one tube with every shared reagent in it, made up at scale and split into the individual reactions, so you pipette the buffer and the primers once instead of thirty times. This calculator works out each reagent volume for the batch from your stock concentrations and the finals your protocol asks for.
It is for anyone setting up a plate or a row of tubes who would rather check the arithmetic than trust a half-remembered dilution. Reactions, volume per reaction, the stocks on the shelf, the finals in the method: that is the whole input.
One honest detail before you start: the fields arrive empty and the tool only prints rows for reagents you give a stock concentration. Forget a final concentration and that reagent drops out silently, which is handy when you are leaving something out and a problem when you are not.
Calculate reagent volumes for a PCR master mix. Enter the number of reactions, per-reaction volumes, and stock concentrations.
How it works
Fill in the number of reactions and the volume per reaction first, because everything else scales from those two: their product is the final volume of the batch. Then come the reagents. 10x buffer needs only a stock, while dNTPs, MgCl2, forward primer, reverse primer and Taq each need a stock and a target final.
The formula is the dilution you learned in first year, volume = (final x total volume) / stock, applied to each reagent with both numbers filled in. Buffer is the exception, using total volume divided by the stock factor because there is no final field: 10x gives a tenth of the batch, always to 1x. Taq is the other, its target being units per reaction rather than a concentration.
Everything comes back in one table of volumes to two decimal places, followed by the total master mix volume and how many reactions it covers. Anything left blank is simply not a row, and Reset clears all thirteen fields and the table.
A worked example
Ten reactions of 25 uL, the two values the form hints at. Total volume is 10 x 25 = 250 uL. A 10x buffer stock gives 250 / 10 = 25.00 uL. dNTPs at 0.2 mM final from a 10 mM stock gives (0.2 x 250) / 10 = 5.00 uL, and MgCl2 at 1.5 mM from 50 mM gives 7.50 uL.
Primers at 0.5 uM final from 10 uM stock give (0.5 x 250) / 10 = 12.50 uL each, so 25.00 uL of primer across both. Taq at 1.25 units per reaction from a 5 units per uL stock gives (1.25 x 10) / 5 = 2.50 uL. The six rows add to 65.00 uL, which leaves 250.00 – 65.00 = 185.00 uL of water, a number the tool does not print.
Twelve reactions instead of ten gives a total of 300 uL, buffer 30.00, dNTPs 6.00, MgCl2 9.00, each primer 15.00, Taq 3.00, a sum of 78.00 uL and 222.00 uL of water. That two reactions of slack is the usual insurance against what stays on the tip.
What the tool does not do
There is no water row and no check that your reagent volumes fit inside the total. You subtract the printed sum yourself, and a mis-entered stock that asks for more volume than you have will still be reported happily. Read the numbers against the total before you touch a pipette.
Buffer always goes to 1x, so a method calling for 0.5x or 2x needs the printed figure halved or doubled by hand. And rows go missing quietly: a blank or zero stock, or a missing final value, is skipped without a message, so a table that looks complete can be missing your reverse primer. Count the rows against your method; it takes ten seconds.
Two decimal places is finer than some pipettes deliver. Scale down to 5 uL reactions and the formula returns hundredths of a microlitre, where anything under about 1 uL wants a dilution of the stock rather than a direct shot into the mix.
The tool only does arithmetic. It does not know whether your polymerase buffer already contains MgCl2, which is the classic route to double the magnesium and a smear instead of a band. Your protocol decides the finals.
Other tools
- Percent yield calculator — the other half of the lab report, once the product is on the balance
- Philippines grades converter — turning the lab mark into the number your transcript shows
- Premed GPA calculator — because the chemistry lab is a four credit course like any other
Frequently asked questions
How much extra master mix should I make?
One or two more than you need, so entering 12 instead of 10 above. The surplus covers what sticks to the tip and the reaction you set up badly, and it is cheaper than repeating a whole plate.
Why is buffer calculated differently?
Because the form asks for a stock but no target final, so the tool assumes 1x. Total volume divided by the stock factor gives a tenth of the batch from a 10x stock and a fifth from a 5x.
What units does Taq take?
Units per reaction on the final field, units per microlitre on the stock field, which is why that row does not use total volume. Typing a concentration into the final field gives a plausible wrong number.
Where does the water go?
It is not in the table. Subtract the printed sum from your total volume and add that much water before you aliquot the mix out.
Why is one of my reagents missing from the table?
Because it has no stock concentration, no final value, or a stock of zero, and those are skipped rather than flagged. Fill in both fields for every reagent you want and the row appears on the next calculation.